Preparation Of Endostatin-loaded Chitosan Nanoparticles And Evaluation Of The Antitumor Effect Of Such Nanoparticles On The Lewis Lung Cancer Model

objective: The purpose of this study was to prepare ES-loaded chitosan nanoparticles(ES-NPs)and evaluate the antiangiogenic and antitumor effect of these particles on Lewis lung cancer model.Methods: ES-NPs were prepared by a simple ionic cross-linking method.The characterization of ES-NPs,including size distribution,loading efficiency(LE)and encapsulation efficiency(EE),was performed.An in vitro release test was used to determine the release behavior of ES-NPs.And the serum stability of ES-NPs at different temperature was also detected.Cell viability and cell migration were assayed to detect the in vitro antiangiogenic effect of ES-NPs.And then,in order to clarify the antitumor effect of ES-NPs in vivo,the Lewis lung cancer models were used.The xenografts were treated by PBS,ES,blank CS nanoparticles and ES-NPs,respectively.During the treatment,the tumor size was measured every two days.Then a tumor growth curve was plotted based on tumor size and length of survival.Mice were sacrificed on day 21 and the tumor tissue and blood simple were collected for immunohistochemistry and ELISA analysis.Results: ES-NPs were successfully synthesized in our study.The ES-NPs made by 40mL(1 mg/mL)CS solution and 500 ?L ES(11.2 mg/mL)showed a suitable size distribution and high EE.The nanoparticles were spherical and homogeneous in shape and exhibited an ideal releasing profile in vitro.Moreover,our nanoparticles could keep stability in serum for at least 48 hours.The result of MTT assay showed that ES-NPs could inhibit the growth of HUVECs.After treatment with ES-NPs for 24 h,the cell viability was slightly reduced.In fact,the result was similar to the viability of cells treated with free ES(p=0.577).However,with longer treatment times,the difference between the ES-NPs and ES groups became significantly larger(p<0.05).For our result of transwell assay,we also found that the migration of HUVECs treated with ES-NPs was significantly inhibited compared to free ES(p<0.05).The in vivo antiangiogenic activity was evaluated by ELISA and immunohistochemistry analyses,which revealed that ES-NPs had a stronger antiangiogenic effect for reinforced anticancer activity.Indeed,even the treatment cycle in which ES-NPs were injected every seven days,showed stronger antitumor effect than the free ES injected for 14 consecutive days.And ES-NPs injected for 14 consecutive days had the best antitumor effect in our study,the tumor growth inhibition rate was high as 63.32%.Conclusion: 1)chitosan is a feasible carrier for endostain to be used in the treatment of tumors.The ES-NPs have high EE and LE,exhibiting an ideal releasing profile in vitro.2)ES-NPs could significantly inhibit the proliferation and migration of HUVECs.And the effect is stronger than that of free ES.3)ES-NPs significantly improved the antitumor activity of ES by affecting angiogenesis.